Lung cancer represents the leading cause of cancers deaths in both men and women worldwide, and current therapies fail to treat this disease in the overwhelming majority of cases. The RAS and p53 pathways are two of the most frequently genetically modified pathways in lung cancers. Alterations in both result in loss of responsiveness to current therapies leading to decreased overall patient survival. Tumor- suppressive microRNA (miRNA) families, including let-7 and miR-34, regulating steps in each of these pathways are often deleted in lung cancer, and their reduced expression is correlated with lung cancer. Objective/Hypothesis: In this work we propose to test the hypothesis that miRNA replacement therapy using miRNAs targeting the RAS pathway with those activated downstream of p53 will represent a novel yet powerful therapeutic to suppress lung tumorigenesis. Specific Aims: (1) To test the hypothesis that modifying let-7 and miR-34 microRNA levels will alter tumor- promoting ability in cell culture. (1a) We will test the effect of overexpression of let-7, miR-34, and the combination in a panel of human lung cancer cell lines with mutations in KRAS, p53 or both. (1b) We will evaluate the ability of anti-miRs to let-7, miR-34, and the combination to transform normal lung epithelial cells. (2) To test the hypothesis that overexpression of let-7 and miR-34 can both prevent and treat an inducible K- ras;p53 mutant mouse model of advanced lung cancer leading to an overall reduction in tumor burden. Study Design: We will address the hypothesis that miRNA treatment can reduce cell growth, invasiveness, and metastatic potential of lung cancer cells with activated KRAS and mutant p53. Cells will be transfected with one of the following: control vector, let-7 expressing vector, mir-34 expressing vector, or let-7/mir-34 expressing vector. Transfected cells will be evaluated by growth curves, ability to form colonies, cell cycle analysis and apoptotic assays such as phoshtidylserine externalization, and caspase activation. In parallel, animals with advanced lung cancer will evaluated for their response to let-7, miR-34 or the combination. For prevention studies, miRNAs will be administered at the same time as Cre recombinase, which induces expression of an activated K-ras allele, and the mutant p53 gene. A separate group of animals will only get Ad-Cre to determine bulk tumor burden in the absence of miRNA treatment. Animals will be sacrificed 8 weeks later, their lungs harvested and therapeutic potential evaluated. In subsequent studies we will evaluate the responsiveness of preformed tumors to miRNAs. Tumor formation will precede treatment by 6 weeks;a time that has been shown to produce early invasive adenocarcinomas in these compound animals. Animals will be sacrificed at 4 weeks post aerosol delivery of lent-let-7, -mir-34, or -let-7/mir-34 and the amount of proliferation in the lungs will be compared to a control group of animals sacrificed at the time of miRNA infection. PUBLIC HEALTH RELEVANCE: Our proposed work on let-7 and miR-34 families of microRNAs has the potential to provide a better understanding of the molecular pathology of lung cancer, knowledge that could lead to significant improvements in lung cancer's treatment and ultimate cure rates in the long term. While this is an emerging field, the benefit to our understanding of miRNAs in cancer will be enormous if we can harness these natural growth repressors as anti-cancer agents